In this Proposal, we will focus on the biology of GVHD and immune regulation by utilizing novel imaging strategies, immune assessments, animal modeling and clinical translational studies to develop a deeper understanding of GVHD and GVT reactions. For the first time, we will explore a novel imaging strategy to image activated T cells, the known effector cells in GVHD, to explore the fate of adoptively transferred cells in the setting of GVHD induction. This approach, if proven to be successful, can also be utilized to explore the biological fate of adoptively transferred T cells such as CAR T cells. We will also explore the biological activity and translational capability of invariant natural killer T (iNKT) cells. Our prior work has demonstrated that iNKT cells have remarkable biological function and are approximately 20-50x more potent than Treg in suppressing GVHD in murine models of HCT. There is also compelling clinical correlative studies to justify intense interest in this cell population. Interestingly, iNKT cells function at very early time points within hours to days following HCT and result in the expansion of Treg highlighting the immune regulatory networks that are critical for proper immune regulation. We will build upon our findings that iNKT cells can be expanded ex vivo and can be used from third party donors with equal efficacy as compared to donor derived cells which is likely to aid in clinical translation. Our goals are to study the biology of iNKT cells in the setting of HCT with the goal of developing an effective strategy to reduce the risk of GVHD while maintaining or enhancing GVT reactions and to develop strategies for their clinical translation in the setting of HCT. We will determine whether in vivo imaging of activated T cells can be successfully imaged to non-invasively identify patients with GVHD and those who are at high risk for developing GVHD with the ultimate goal of earlier intervention that may be more effective. We will test whether invariant NKT cells result in the expansion of a population of Treg with enhanced function resulting in prevention of GVHD and can be engineered to maintain or enhance GVL activities. We will test the hypothesis that iNKT cells hold great promise in reducing the risk of GVHD while maintaining GVT reactions and through the proposed studies our goal is to develop strategies to isolate and expand donor or third party iNKT cells for GVHD prevention and improvement in GVHD free, relapse free survival.